Recent years have seen rapid progress in research on the use of drugs based on oligonucleotides, which are relatively low molecular nucleic acids. Particular attention is being directed toward antisense sequences that inhibit translation of genetic information by forming complementary double helixes with mRNA, triple-helix-forming oligonucleotides, or TFOs, that form triple helixes (triplexes) with chromosomal gene sequences and thereby inhibit transcription of genetic information, and decoys that inhibit transcription of genetic information by sequence-specific binding to nucleic acid-binding proteins such as transcription factors. However, the specific binding of such oligonucleotide drugs to their targets is known to be often weak and relatively prone to dissociation. A high demand therefore exists for development of new oligonucleotide drugs capable of binding more strongly to their targets, so that the oligonucleotides can be more effectively used as drugs.
The possibility of introducing point mutations into gene sequences by base sequence-specific crosslinking reaction has recently been noted (Woolf, T. et al., Nature Biotech. 1998, 16, 341), and there has been considerable focus on possible applications thereof. Numerous reports have therefore appeared with the goal of introducing reactive groups into oligonucleotides to achieve a target sequence-specific crosslinking reaction(s). Examples of such reactive groups include haloacetamides (Grant, K. et al., Biochemistry 1.996, 65, 12313), aziridines (Shaw, J. et al., J. Am. Chem. Soc. 1991, 113, 7765) and psoralen derivatives (Chang, E. et al., Biochemistry 1991, 30, 8283), which have been the subject of much investigation. However, non-specific bond formation tends to occur with these reactive groups and this has prevented achieving a truly target-specific crosslinking reaction(s).
While attempting to solve the problems mentioned above, the present inventors have previously found that the 2-amino-6-vinylpurine structure undergoes a base sequence-specific crosslinking reaction with cytidine (Nagatsugi, F. et al., Tetrahedron 1997, 53, 3035). However, the 2-amino-6-vinylpurine structure is not suitable because of its extremely high reactivity, which tends to produce a non-specific reaction(s) with the amino groups in proteins.
While attempting to solve this problem, the present inventors also found that it is useful to obtain phenylsulfide derivatives and phenylsulfoxide derivatives at the vinyl group of 2-amino-6-vinylpurine (Nagatsugi, F. et al., J. Am. Chem. Soc. 1999, 121, 6753). These derivatives are oxidized to produce the highly reactive 2-amino-6-vinylpurine after hydrogen bonding-driven formation of a complex with cytidine which has the target and complementary structure. The stability is high and non-specific reaction is minimized. In addition, through synthesis of an amidite reagent comprising the phenylsulfide derivative of the 2-amino-6-vinylpurine structure and its use as a reagent for oligonucleotide synthesis using a DNA synthesizer, it has become possible to introduce the phenylsulfide derivative of 2-amino-6-vinylpurine as a reactive group at any desired location of an oligonucleotide. Oxidation of the phenylsulfide derivative will then accomplish introduction of the phenylsulfoxide derivative of 2-amino-6-vinylpurine at any desired location of the oligonucleotide. However, the low reactivity of phenylsulfide derivative-containing oligonucleotides with their target nucleic acids remains a problem.